Quite frequently in biomedical research there is a need to detect and monitor dynamic chemistries in solution in the vicinity of an interface. For instance, many studies focus on detection of chemical secretions from cultured tissues or cells into the surrounding medium. Often, such applications place specific demands on the required spatial and temporal resolution of the detection method. When monitoring secretions this would be due to heterogeneity in the cell types and behavior, and also variation in cellular activities with time. Labeling, with, for instance, a fluorescent marker, a radioactive marker, or using antigen/ antibody attachment, has been spectacularly successful as the foundation for imaging dynamic biochemistry, but concerns about the altering of labeled analyte behavior and non-specific binding cannot be eliminated. Furthermore, all targeted methods, including those based on labeling, are inherently limited in their discovery potential, as one cannot find what one is not looking for. The purpose of the proposed research is to overcome the inherent limitations of current biochemical imaging technologies. This will be done through the development of electrospray ion sources that can serve as mass spectrometry probes (MSP) for highly resolved biochemical detection from the microenvironment adjacent to biological interfaces. The research team has a demonstrated history of success inventing novel mass spectrometry ion sources, and proposes, for this project, to accomplish the ambitious task of combining all prerequisite capabilities for sample collection, processing, and ionization into a micro- sampling capillary. This "lab-on-a-tip" will include in-line microdialysis to remove salts and exchange solvent, as well as an integrated tryptic digestion micro-reactor. The research team will develop, optimize and demonstrate MSP through an established multifaceted approach combining experiment (including optical and mass spec characterization), analysis and simulation (first principles physical models and computational fluid dynamics), and state of the art manufacturing (microfabrication). MSP will assume an important role in biological research as a hypothesis generator, and will become a key tool in improving development of bioreactors for regenerative medicine applications. Successful results have potential for transformational benefits to a wide range of research applications, including biomarker discovery, improved understanding of healthy and diseased cell biology, biosensor development, and bio-manufacturing process analysis and control. In addition to presentation at conferences and publication in archival journals, the application of MSP technology to biological problems will be disseminated through an educational workshop hosted at Ga. Tech. Furthermore, the probe will be coupled to a TOF mass spectrometer that is part of the NSF supported National Nanotechnology Infrastructure Network (NNIN), and therefore available to users from industry and academic institutions alike. PUBLIC HEALTH RELEVANCE (provided by applicant): In this application, we propose to develop an ambient Mass Spectrometry Probe (MSP), which passively samples and softly ionizes large biomolecules directly from liquid at physiological conditions, and thus makes possible transient mass spectrometric monitoring at precisely controlled locations in a complex liquid environment, with MS spectra collected as a function of time. The resulting capability to directly monitor biochemistry in physiologically relevant solution conditions will enable biologists, biochemists, and bioengineers to spatially correlate chemical data with high precision, not only for the generation of clear chemical "images" of in vitro cultures, but also for the investigation of the role of heterogeneity and gradients in numerous important applications, including bio reactors for regenerative medicine, activity and behavior of bioengineered materials, and biosensor characterization. MSP will assume an important role in biological research as a hypothesis generator, and has the potential for transformational benefits to a wide range of research endeavors, including biomarker discovery, investigations of healthy and diseased cell biology, biosensor development, and bio-manufacturing process analysis and control.